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We are recently faced with a progressive evolution of the therapeutic paradigm for radioiodine refractory differentiated thyroid cancer (RAI-R DTC), since the advent of tissue agnostic inhibitors. Thus, tumor genotype assessment is always more relevant and is playing a crucial role into clinical practice. We report the case of an elderly patient with advanced papillary thyroid carcinoma (PTC) harboring RET-CCDC6 fusion with four co-occurring mutations involving PI3KCA, TP53, and hTERT mutations, treated with pralsetinib under a compassionate use program. Despite the high histological grade and the coexistence of aggressive RET co-mutations, an impressive metabolic and structural tumor response has been obtained, together with a patient’s prolonged clinical benefit. A timely comprehensive molecular testing of those cases wild-type for the common thyroid carcinoma BRAF V600E-like and RAS-like driver mutations may uncover actionable gene rearrangements that can be targeted by highly selective inhibitors with great potential benefit for the patients.

The widespread use of more sensitive detection tools, such as next-generation sequencing, has increased the identification of a variety of BRAF mutations other than V600E/K in melanoma patients. However, there is a lack of established data regarding the efficacy of BRAF/MEK inhibitors and immune-checkpoint immune inhibitors (ICI) for these patients. We performed a retrospective study, including all the patients diagnosed with stage III or IV melanoma that were referred to the University Hospital of Bologna from 2011 to 2021, carrying a non-V600E or V600K mutation of BRAF and who were started on systemic treatment. We found 14 patients with stage III or IV melanoma harboring the following BRAF mutations: V600R, V600_K601delinsE, K601E, p.T599_V600insT, L597V, G466R, S467L, and A598T. Of note, G466R and A598T BRAF mutations have never been previously reported in melanoma. Four patients received combined BRAF/MEK inhibitors, two patients BRAF inhibitor monotherapy, and six patients were treated with ICI for advanced melanoma; four patients received adjuvant treatment with nivolumab. Given the few cases and the absence of randomized clinical trials, it is important to report clinical experiences, which can guide physicians in the treatment of melanomas harboring rare BRAF mutations.

(1) Background: Human papillomaviruses (HPVs) are known to be related to the development of about 5% of all human cancers. The clinical relevance of HPV infection has been deeply investigated in carcinomas of the oropharyngeal area, uterine cervix, and anogenital area. To date, several different methods have been used for detecting HPV infection. The aim of the present study was to compare three different methods for the diagnosis of the presence of the HPV genome. (2) Methods: A total of 50 samples were analyzed. Twenty-five of them were tested using both next generation sequencing (NGS) and VisionArray® technology, the other 25 were tested using Hybrid Capture (HC) II assay and VisionArray® technology. (3) Results: A substantial agreement was obtained using NGS and VisionArray® (κ = 0.802), as well as between HC II and VisionArray® (κ = 0.606). In both analyses, the concordance increased if only high risk HPVs I(HR-HPVs) were considered as “positive”. (4) Conclusions: Our data highlighted the importance of technical choice in HPV characterization, which should be guided by the clinical aims, costs, starting material, and turnaround time for results.

BACKGROUND Non-canonical isocitrate dehydrogenase (IDH) mutations are uncommon among patients with grade 2 and 3 gliomas and their clinical significance is still unclear. METHOD We carried out a systematic review and meta-analysis to evaluate the prognostic impact of IDH1 and IDH2 non-canonical mutations. We searched English-written articles published on PubMed/Medline, Cochrane library, and Scopus until the 1st May 2021. Keywords adopted for the research were: ‘’IDH’’ OR ‘’IDH1’’ OR ‘’IDH2’’ OR ‘’Isocitrate dehydrogenase’’ AND ‘’glioma’’. RESULTS We selected 13 of 3513 studies reporting data of 4007 patients with a diagnosis of grade 2 and grade 3 including 3091 patients with a molecular proven IDH 1 or IDH 2 mutations. Between patients with IDH mutated gliomas, 479 (15.5%) patients showed non-canonical IDH 1 or IDH 2 mutations. The presence of IDH 1 non-canonical mutation occurred in younger age and in non codeleted 1p19q as compared to canonical IDH 1 mutation. However, patients with IDH 2 mutations showed more frequently 1p19q codeletion as compared to those with canonical IDH 1 mutated glioma. Four studies reported complete data survival for patients with non-canonical (n= 160) and canonical mutations (n= 1019). The pooled HR of these studies was 0.47 (95% CI, 0.28-0.81) confirming a positive prognostic role for non-canonical IDH 1 and IDH 2 mutations. CONCLUSION The presence of non-canonical IDH 1 and IDH 2 mutations defines a specific subgroup of gliomas occurring at younger age with improved survival. Patients with a non-canonical IDH 1 mutation showed less frequently 1p19q codeletion as compared to those patients harboring a canonical or IDH 2 mutation.

IntroductionThe Cancer Genome Atlas (TCGA) project identified four distinct prognostic groups in endometrial cancer (EC), among which two are correlated with an intermediate prognosis: the MisMatch Repair-deficient (MMRd) and the No Specific Molecular Profile (NSMP) groups. The two groups represent a heterogeneous subset of patients frequently harboring CTNNB1 alterations with distinctive clinicopathologic features. The study aimed to evaluate the miRNA expression in ECs to identify potential biomarkers of prognosis.MethodsWe analyzed miRNA expression in 72 ECs classified as MMRd or NSMP including 15 ECs with CTNNB1 mutations. In the discovery step, miRNA expression was evaluated in 30 cases through TaqMan miRNA arrays. Subsequently, four miRNAs were validated in the total cohort of ECs. The data were further tested in the TCGA cohort, and correlations with overall survival (OS) and progression-free interval (PFI) were evaluated.ResultsmiR-499a-3p and miR-499a-5p resulted to be overexpressed in CTNNB1 mutant EC patients at intermediate risk. Similarly, in the TCGA cohort, miR-499a-3p and miR-499a-5p were differentially expressed between CTNNB1 mutant and wild-type patients (p < 0.0001). NSMP patients with low miR-499a-5p expression showed longer OS (p = 0.03, log-rank test). By combining miR-499a-3p or -5p expression levels with the CTNNB1 status, ECs with CTNNB1 mutation and lower miR-499a-5p expression showed better OS compared with the other subgroups (p = 0.03, log-rank test), among the NSMP patients. Moreover, in a multivariate analysis, combination of wild type CTNNB1 status and high miR-499a-5p expression was indipendently associated with high risk of death [HR (95%CI): 3.53 (1.1-10.5), p = 0.02].ConclusionOur results suggest that the combination of CTNNB1 status and miR-499a-5p allows a better stratification of NSMP patients and could promote a personalization of the treatment in intermediate-risk patients.

Endometrial cancer (EC) is the most common gynecological cancer, with annual incidence rates in Western countries ranging between 15 and 25 per 100.000 women. About 15-20% of patients with EC have high-risk disease and follow an aggressive clinical course. Unfortunately, the assessment of histologic parameters is poorly reproducible and conventional clinicopathological and molecular features do not reliably predict either the patient's response to the available treatments or the definition of personalized therapeutic approaches. In this context, the identification of novel diagnostic and prognostic biomarkers, which can be integrated in the current classification schemes, represents an unmet clinical need and an important challenge. MiRNAs are key players in cancer by regulating the expression of specific target genes. Their role in EC, in association with clinical and prognostic tumor biomarkers, has been investigated but, so far, with little consensus among the studies. The present review aims to describe the recent advances in miRNAs research in EC taking into consideration the current classification schemes and to highlight the most promising miRNAs. Finally, a perspective point of view sheds light on the challenges ahead in the landscape of endometrial cancer. This article is protected by copyright. All rights reserved.

Immunotherapy targeting the PD-1–PD-L1 axis yielded good results in treating different immunologically ‘‘hot’’ tumors. A phase II study revealed good therapeutic activity of pembrolizumab in selected prostatic carcinoma (PC)-patients. We performed a systematic literature review (PRISMA guidelines), which analyzes the immunohistochemical expression of PD-L1 in human PC samples and highlights the pre-analytical and interpretation variables. Interestingly, 29% acinar PCs, 7% ductal PCs, and 46% neuroendocrine carcinomas/tumors were PD-L1+ on immunohistochemistry. Different scoring methods or cut-off criteria were applied on variable specimen-types, evaluating tumors showing different clinic-pathologic features. The positivity rate of different PD-L1 antibody clones in tumor cells ranged from 3% (SP142) to 50% (ABM4E54), excluding the single case tested for RM-320. The most tested clone was E1L3N, followed by 22C3 (most used for pembrolizumab eligibility), SP263, SP142, and 28-8, which gave the positivity rates of 35%, 11–41% (depending on different scoring systems), 6%, 3%, and 15%, respectively. Other clones were tested in

Background: Non-canonical mutations of the isocitrate dehydrogenase (IDH) genes have been described in about 20–25% and 5–12% of patients with WHO grade II and III gliomas, respectively. To date, the prognostic value of these rare mutations is still a topic of debate. Methods: We selected patients with WHO grade II and III gliomas and IDH1 mutations with available tissue samples for next-generation sequencing. The clinical outcomes and baseline behaviors of patients with canonical IDH1 R132H and non-canonical IDH1 mutations were compared. Results: We evaluated 433 patients harboring IDH1 mutations. Three hundred and ninety patients (90.1%) had a canonical IDH1 R132H mutation while 43 patients (9.9%) had a non-canonical IDH1 mutation. Compared to those with the IDH1 canonical mutation, patients with non-canonical mutations were younger (p &lt, 0.001) and less frequently presented the 1p19q codeletion (p = 0.017). Multivariate analysis confirmed that the extension of surgery (p = 0.003), the presence of the 1p19q codeletion (p = 0.001), and the presence of a non-canonical mutation (p = 0.041) were variables correlated with improved overall survival. Conclusion: the presence of non-canonical IDH1 mutations could be associated with improved survival among patients with IDH1 mutated grade II–III glioma.

Simple Summary Vulvar squamous cell carcinoma (VSCC) is the most frequent malignant vulvar tumor, with a peak incidence in the 7–8th decades of life. However, VSCCs can also occur in young women. This unfortunate event is even rarer and more worrisome in pregnant women, being hard to manage for gynecologists, oncologists, and radiotherapists. Very few cases have been reported and we felt the need for an updated review on this topic. Thus, we performed a systematic literature review of VSCCs diagnosed during pregnancy, discussing the clinic-pathologic features, the implications in pregnancy outcomes, and the effects of such a diagnosis in the management of mothers and their babies. Abstract According to our systematic literature review (PRISMA guidelines), only 37 vulvar squamous cell carcinomas (VSCCs) were diagnosed during pregnancy (age range: 17–41 years). The tumor size range was 0.3–15 cm. The treatment was performed after (14/37, 38%), before (10/37, 27%), or before-and-after delivery (11/37, 30%). We found that 21/37 (57%) cases were stage I, 2 II (5%), 11 III (30%), and 3 IVB (8%). HPV-related features (condylomas/warts; HPV infection; high-grade squamous intraepithelial lesion) were reported in 11/37 (30%) cases. We also found that 9/37 (24%) patients had inflammatory conditions (lichen sclerosus/planus, psoriasis, chronic dermatitis). The time-to-recurrence/progression (12/37, 32%) ranged from 0 to 36 (mean 9) months. Eight women died of disease (22%) 2.5–48 months after diagnosis, 2 (5%) were alive with disease, and 23 (62%) were disease-free at the end of follow-up. Pregnant patients must be followed-up. Even if they are small, newly arising vulvar lesions should be biopsied, especially in women with risk factors (HPV, dermatosis, etc.). The treatment of VSCCs diagnosed in late third trimester might be delayed until postpartum. Elective cesarean section may prevent vulvar wound dehiscence. In the few reported cases, pregnancy/fetal outcomes seemed to not be affected by invasive treatments during pregnancy. However, clinicians must be careful; larger cohorts should define the best treatment. Definite guidelines are lacking, so a multidisciplinary approach and discussion with patients are mandatory.

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Background A synonymous single nucleotide polymorphism (SNP) is a substitution of a single base that does not modify the primary amino acid sequence but could influence protein function. In patients with brain tumors, the incidence of the silent SNP IDH 1 105GGT (rs11554137) is three times higher than the normal population. Methods Our aim was to investigate the prognostic role of the IDH 1 105GGT SNP. We selected only patients with diagnosis of IDH grade II or III mutated glioma. Additional inclusion criteria were: complete clinical data and adequate tumor samples for IDH 1 or 2 sequencing. Results 71 patients with grade II and III IDH-mutated glioma have been evaluated. Nine of 71 patients (12.7 %) presented the SNP 105GGT. Patients with SNP 105GGT had a longer Progression Free Survival (PFS - 47.3 months vs Not reached; p = 0.015). The SNP 105GGT (HR 0.240; 95 %CI 0.074−0.784, p = 0.018) was confirmed as an independent prognostic factors in multivariate analysis. Conclusions Patients with IDH1 or 2 mutated grade II and III glioma presenting the SNP105GGT had longer PFS regardless adjuvant treatment received and extension of primary surgery. A validation is warranted to confirm our preliminary results.

No abstract available

Many studies have investigated the potential prognostic and predictive role of PD-L1 in prostatic carcinoma (PC). We performed a systematic literature review (PRISMA guidelines) to critically evaluate human tissue-based studies (immunohistochemistry, molecular analysis, etc.), experimental research (cell lines, mouse models), and clinical trials. Despite some controversial results and study limitations, PD-L1 expression by tumor cells may be related to clinic–pathologic features of adverse outcome, including advanced tumor stage (high pT, presence of lymph node, and distant metastases), positivity of surgical margins, high Grade Group, and castration resistance. Different PD-L1 positivity rates may be observed in matched primary PCs and various metastatic sites of the same patients. Over-fixation, type/duration of decalcification, and PD-L1 antibody clone may influence the immunohistochemical analysis of PD-L1 on bone metastases. PD-L1 seemed expressed more frequently by castration-resistant PCs (49%) as compared to hormone-sensitive PCs (17%). Some series found that PD-L1 positivity was associated with decreased time to castration resistance. Treatment with ipilimumab, cyclophosphamide/GVAX/degarelix, or degarelix alone may increase PD-L1 expression. Correlation of PD-L1 positivity with overall survival and outcomes related to tumor recurrence were rarely investigated; the few analyzed series produced conflicting results and sometimes showed limitations. Further studies are required. The testing and scoring of PD-L1 should be standardized.

The collaborative Cancer Genome Atlas (TCGA) project identified four distinct prognostic groups of endometrial carcinoma (EC) based on molecular alterations: (i) the ultramutated subtype that encompasses POLE mutated (POLE) cases, (ii) the hypermutated subtype, characterized by MisMatch Repair deficiency (MMRd), (iii) the copy-number high subtype, with p53 abnormal/mutated features (p53abn), (iv) the copy-number low subtype, known as No Specific Molecular Profile (NSMP). Although the prognostic value of TCGA molecular classification, NSMP carcinomas present a wide variability in molecular alterations and biological aggressiveness. This study aims to investigate the impact of ARID1A and CTNNB1/β-catenin alterations by targeted Next-generation sequencing (NGS) and immunohistochemistry (IHC) in a consecutive series of 125 molecularly classified ECs. NGS and IHC were used to assign surrogate TCGA groups and to identify molecular alterations of multiple target genes including POLE, PTEN, ARID1A, CTNNB1, TP53. Associations with clinicopathologic parameters, molecular subtypes, and outcomes identified NSMP category as the most heterogeneous group in terms of clinicopathologic features and outcome. Integration of surrogate TCGA molecular classification with ARID1A and β-catenin analysis showed NSMP cases with ARID1A mutation characterized by the worst outcome with early recurrence, while NSMP tumors with ARID1A wild-type and β-catenin alteration had indolent clinicopathologic features and no recurrence. This study indicates how the identification of ARID1A and β-catenin alterations in EC represents a simple and effective way to characterize NSMP tumor aggressiveness and metastatic potential.

AimsIn metastatic colorectal carcinomas (mCRC), RAS/RAF genes mutations are first tested to determine the eligibility for anti-EGFR (Epidermal Growth Factor Receptor) therapy in combination with conventional cytotoxic agents. Recent advancements in next-generation sequencing (NGS) have highlighted the potential of multi-gene panels. This multi-gene analysis may provide useful information for the molecular characterisation of mCRC, other than the status of RAS/RAF genes. Aim of this study was to evaluate the feasibility of two NGS custom multi-gene panels in the characterisation of CRC cases and evaluating the relevance of PIK3CA mutation in a routine cohort of consecutive CRC cases.MethodsA total of 961 formalin-fixed and paraffin-embedded specimens from two medical centres (Bologna and Naples) were analysed using two lab-developed NGS multi-gene panels.ResultsKRAS mutations (56.2%) were the more frequent alterations observed in our cohort. Intriguingly, PIK3CA mutations were more frequent (16.8%) than variants observed in the other two genes nowadays analysed in CRC clinical practice (NRAS and BRAF, 4.2% and 9.6%, respectively). Moreover, in more than 10% of samples, coexistent mutations were detected in our cohort of CRC.ConclusionsOur study demonstrates the feasibility and efficacy of lab-developed targeted multi-gene NGS panels in the clinical practice of CRC. Moreover, the data lead to hypothesise that PIK3CA mutations, together with those of RAS/BRAF, worth to be further investigated in clinical CRC specimens.

Medulloblastoma is a highly aggressive brain tumor that typically affects children, while in adults it represents ~1% of all brain tumors. Little is known about microRNA expression profile of the rare adult medulloblastoma. The main aim of this study was to identify peculiar differences in microRNA expression between childhood and adult medulloblastoma. Medulloblastomas were profiled for microRNA expression using the Exiqon Human miRNome panel (I + II) analyzing 752 microRNAs in a training set of six adult and six childhood cases. Then, the most differentially expressed microRNAs were validated in a total of 21 adult and 19 childhood cases. Eight microRNAs (miR-196b-5p, miR-183-5p, miR-200b-3p, miR-196a-5p, miR-193a-3p, miR-29c-3p, miR-33b-5p, and miR-200a-3p) were differentially expressed in medulloblastoma of adults and children. Analysis of the validation set confirmed that miR-196b-5p and miR-200b-3p were significantly overexpressed in medulloblastoma of adults as compared with those of children. We followed an in silico approach to investigate direct targets and the pathways involved for the two microRNAs (miR-196b and miR-200b) differently expressed between adult and childhood medulloblastoma. Adult and childhood medulloblastoma have different miRNA expression profiles. In particular, the differential dysregulation of miR-196b-5p and miR-200b-3p characterizes the miRNA profile of adult medulloblastoma and suggests potential targets for novel diagnostic, prognostic, or therapeutic strategies.

Anaplastic thyroid cancer (ATC) is a rare but highly aggressive form of thyroid cancer. By contrast, differentiated papillary thyroid cancer (PTC) only rarely behave aggressively and develop distant metastasis. Whether distantly metastatic PTC (DM-PTC) and ATC share a common genetic background is still to be defined. We used next-generation sequencing (NGS) to explore the genetic background of a cohort of ATC and DM-PTC and a group of well-differentiated PTCs that did not developed distant metastasis as control (ctrl-PTC). A panel of 128 amplicons within 21 thyroid cancer-related genes was analyzed in a set of 151 thyroid cancer samples including 66 ATCs and DM-PTCs. We showed that the ATC/DM-PTC group had an overall mutational load higher than ctrl-PTCs and that ATCs and DM-PTCs are characterized by a different genetic background, with the exception of mutations in the TERT promoter that were overrepresented in both ATCs (61.1%) and DM-PTCs (48.2%) vs non-aggressive ctrl-PTCs (7.6%). In ATCs, TERT promoter mutations were frequently associated with TP53 mutations, while in the DM-PTCs no significant co-occurrence was observed. No significant association of MED12 mutations with aggressiveness of thyroid cancer was observed in our analysis. Finally, correlation analysis showed that increasing number of mutations negatively impact on patient overall survival also within the ATC and DM-PTC group. In conclusions, overall our analysis further highlights the relevance of TERT promoter mutations in driving aggressiveness and provides new pieces of information in the definition of aggressiveness evolution of thyroid cancer lesions.

Aim: To evaluate relevance of clinical and molecular factors in adult low-grade gliomas (LGG) and to correlate with survival. Methods: We reviewed records from adult LGG patients from 1991 to 2015 who received surgery and had sufficient tissue to molecular biomarkers characterization. Results: 213 consecutive LGG patients were included: 17.4% were low-risk, according to Radiation Therapy Oncology Group (RTOG) risk assessment. IDH 1/2 mutation, 1p/19q co-deletion, MGMT methylation were found in 93, 50.8 and 65.3% of patients. Median follow-up was 98.3 months. In univariate analysis, overall survival was influenced by extent of resection (p = 0.011), IDH mutation (p

Malignancies consist not only of cancerous and nonmalignant cells, but also of additional elements, as extracellular matrix. The aim of this review is to summarize meta-analyses, describing breast tissue stiffness and risk of breast carcinoma (BC) assessing the potential relationship between matricellular proteins (MPs) and survival. A systematic computer-based search of published articles, according to PRISMA statement, was conducted through Ovid interface. Mammographic density and tissue stiffness are associated with the risk of BC development, suggesting that MPs may influence BC prognosis. No definitive conclusions are available and additional researches are required to definitively clarify the role of each MP, mammographic density and stiffness in BC development and the mechanisms involved in the onset of this malignancy.

According to the 2016 World Health Organization (WHO) classification of tumors of the central nervous system, assessment of exon 4 mutations in isocitrate dehydrogenase 1 or 2 genes (IDH1 or IDH2) is an essential step in the characterization of gliomas. The p.R132H mutation is the most frequent alteration in IDH genes, however other non-canonical IDH mutations can be identified. The aim of this study is to investigate in depth the prevalence of non-R132H IDH ("non-canonical") mutations in brain tumors classified according to the 2016 WHO scheme and their clonal distribution in neoplastic cells. A total of 288 consecutive cases of brain gliomas (grade II-IV) were analyzed for exon 4 IDH1 and IDH2 mutations. IDH1 and IDH2 analysis was performed using next generation sequencing. Non-canonical IDH mutations were identified in 13/52 (25.0%) grade II gliomas (astrocytomas: 8/31, 25.8%; oligodendrogliomas: 5/21, 23.8%) and in 5/40 (12.5%) grade III gliomas (astrocytomas: 3/25, 12.0%; oligodendrogliomas: 2/15, 13.3%). They were not identified in 196 grade IV gliomas (192 glioblastomas, 4 gliosarcomas). In the large majority (>80%) of tumors IDH mutations, both IDH1-R132H and the non-canonical ones, were present in the large majority (>80%) of neoplastic cells. Our data highlight the importance of investigating not only the IDH1-R132H mutation but also the non-canonical ones. These mutations are clonally distributed, with proportions of mutated neoplastic cells overlapping with those of p.R132H, a finding consistent with their driver role in gliomagenesis.

BACKGROUND Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. METHODS Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. RESULTS All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization–time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. CONCLUSIONS Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n=10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Aim: European Organization for Research and Treatment of Cancer (EORTC) and the Radiation Therapy Oncology Group (RTOG) criteria are used to choose treatment in low-grade gliomas. However, no data exist on their concordance. Methods: Low-grade glioma patients treated at our institution from 1998 to 2015 and assessable for both RTOG and EORTC criteria were included to analyze their concordance. Surgery extension, postsurgical treatments, molecular characteristics ( IDH mutation, MGMT methylation and 1p/19q codeletion) were recorded. Results: We included 99 patients. The concordance was low (50.5%; K = 0.127; p = 0.021) but for two subgroups: EORTC high-risk patients were also RTOG high-risk patients (concordance: 97.5%) and RTOG low-risk patients were also EORTC low-risk patients (concordance: 90.9%). Conclusion: The concordance between RTOG and EORTC criteria is low. Thus, clinical trials adopting different risk criteria are not comparable.

e20515 Background: Large cell neuroendocrine carcinoma of the lung (LCNEC) is a rare tumor, with clinical and molecular features between non-small cell and small-cell lung cancer (NSCLC and SCLC, respectively). To date, little is known about factors able to predict clinical outcome of these patients. We sought to identify prognostic factors in LCNEC. Methods: We retrospectively collected pathologically-confirmed LCNEC patients treated at the S. Orsola Malpighi Hospital of Bologna between 01/01/2009 and 31/12/2020. LCNEC was defined as tumor with neuroendocrine morphology, expression of ≥2 neuroendocrine markers (including chromogranin A, synaptophysin, or CD56) at immunohistochemistry and high tumor grade (defined as necrosis and high Ki67 ≥ 50% or high mitotic rate, ≥ 10 mitoses per 2 mm2). Clinical features, pathological features, and blood test values (including neuron-specific enolase [NSE], carcinoembryonic antigen [CEA], cytokeratin 19 fragment [cyfra 21-1]) were correlated with progression-free survival to first-line chemotherapy (PFS) and overall survival from the diagnosis of extensive disease (OS). Results: Eighty-one LCNEC patients (median age 69 years, range 63-76) were identified. Twenty-seven (33.3%) had limited-stage disease and 52 patients (67.5%) had Eastern cooperative oncology group performance status (ECOG PS) ≥ 1 at diagnosis. When extensive disease was found (ED-LCNEC, N = 67), 23 patients (34.3%) had liver metastases, 10 (14.9%) had brain metastases and 24 (35.8%) had bone metastases. Treatment in ED-LCNEC was platinum plus etoposide in 45 patients (81.9%) and platinum plus paclitaxel in 10 patients (18.1%). Among 55 patients treated with platinum-based chemotherapy, 5 patients (9.1%) received cisplatin, while 50 (90.9%) received carboplatin. Overall, OS was 9.17 months (95% confidence interval [95%CI]7.17-13.87), PFS was 4.87 months (95%CI 4.21-6.02) and ORR was 38.2% (95%CI 25.4-42.3, N = 21/56). At univariate analysis, ECOG PS ≥ 1 (P = 0.049), presence of liver (P = 0.004) and bone metastases (P = 0.009), ≥ 2 non-nodal metastatic sites (P < 0.001), elevated NSE (P = 0.009) and CEA level at diagnosis (P = 0.011) were associated with increased risk of death. At multivariate analysis, ECOG PS ≥ 1 (P = 0.020), ≥ 2 metastatic sites (P = 0.002), and elevated NSE level at diagnosis (P = 0.009) were independently associated with the risk of death. At univariate analysis brain metastases (P = 0.017), ≥2 metastatic sites (P = 0.006) and elevated value of CEA at baseline (P = 0.026) were associated with increased risk of progression. At multivariate analysis, only the presence of brain metastases (P = 0.043) retained its association with the risk of progression. Conclusions: In ED-LCNEC of the lung, ECOG PS, number of non-nodal sites of metastases and NSE at baseline are associated with worse survival, while the presence of brain metastases is associated with shorter PFS.

Clinical Practice Points • p.E749Q is an uncommon EGFR mutation in exon 19 with unknown clinical significance. • A patient with lung adenocarcinoma harboring the p.E749Q EGFR mutation does not respond to gefitinib. • The p.E749Q mutation seems to be correlated with resistance to tyrosine kinase inhibitors. • Conventional chemotherapy should be the treatment of choice in patients with the p.E749Q mutation.

BRAF exon 15 mutations are the most common molecular alterations found in papillary thyroid carcinoma (PTC). To date, there is no information regarding BRAF alterations in the thyroid parenchyma surrounding the tumor. To explore the early events associated with the development of PTC, we used massively parallel sequencing to investigate BRAF exon 15 in 30 PTCs and in 100 samples from the thyroid parenchyma surrounding the tumor. BRAF p.V600E was identified in 19/30 PTCs (63.3%). BRAF p.V600E mutations were identified in the tissue adjacent the PTC only in samples containing psammoma bodies. The other samples were either BRAF wild type (WT) or carried BRAF non p.V600E mutations. Specifically, BRAF p.G593D, -p.A598T, -p.V600M, -p.R603Q, -p.S607F, and -p.S607P were identified in 4 of 36 (11.1%) samples with follicular cell atypia, in 2 of 16 (12.5%) with follicular cell hyperplasia, and in 1 of 33 (3.0%) histologically normal samples&mdash, only in tissue surrounding BRAF p.V600E mutated PTCs. These mutations are predicted to affect protein function in silico but, in vitro, have kinase activity and BRAF phosphorylation levels similar to BRAF WT. No BRAF exon 15 mutations were identified in samples adjacent to PTCs that were BRAF WT. A mutagenic process affecting BRAF exon 15 occurs in a subset of thyroid glands that develop BRAF p.V600E mutated PTCs.

It is unclear whether the site of origin of papillary thyroid microcarcinoma (mPTC) with respect to the thyroid surface has an influence on clinicopathologic parameters. The objectives of the study were to: (i) Accurately measure the mPTC distance from the thyroid surface, (ii) analyze whether this distance correlates with relevant clinicopathologic parameters, and (iii) investigate the impact of the site of origin of the mPTC on risk stratification. Clinicopathologic features and BRAF mutational status were analyzed and correlated with the site of origin of the mPTC in a multicenter cohort of 298 mPTCs from six Italian medical institutions. Tumors arise at a median distance of 3.5 mm below the surface of the thyroid gland. Statistical analysis identified four distinct clusters. Group A, mPTC: size &ge, 5 mm and distance of the edge of the tumor from the thyroid capsule = 0 mm, group B, mPTC: size &ge, 5 mm and distance of the edge of the tumor from the thyroid capsule &gt, 0 mm, group C, mPTC: size &lt, and group D, mPTC: size &lt, 0 mm. Univariate analysis demonstrates significant differences between the groups: Group A shows the most aggressive features, and group D the most indolent ones. By multivariate analysis, group A tumors are characterized by tall cell histotype, BRAF V600E mutation, tumor fibrosis, aggressive growth with invasive features, vascular invasion, lymph node metastases, and intermediate ATA risk. The mPTC clinicopathologic features vary according to the tumor size and distance from the thyroid surface. A four-group model may be useful for risk stratification and to refine the selection of nodules to be targeted for fine needle aspiration.

// Greta Gandolfi 1 , Moira Ragazzi 2 , Dario de Biase 3 , Michela Visani 4 , Eleonora Zanetti 2 , Federica Torricelli 1 , Valentina Sancisi 1 , Mila Gugnoni 1 , Gloria Manzotti 1 , Luca Braglia 5 , Silvio Cavuto 5 , Domenico Franco Merlo 5 , Giovanni Tallini 4 , Andrea Frasoldati 6 , Simonetta Piana 2 and Alessia Ciarrocchi 1 1 Laboratory of Translational Research, Azienda Unita Sanitaria Locale di Reggio Emilia - IRCCS, Reggio Emilia 42123, Italy 2 Pathology Unit, Department of Oncology, Azienda Unita Sanitaria Locale di Reggio Emilia - IRCCS, Reggio Emilia 42123, Italy 3 Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, 40139 Bologna, Italy 4 Department of Medicine, Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale-DIMES, Anatomic Pathology Unit, Bellaria Hospital, University of Bologna, 40139 Bologna, Italy 5 Research and Statistics Unit, Azienda Unita Sanitaria Locale di Reggio Emilia-IRCCS, Reggio Emilia, 42123, Italy 6 Endocrinology Unit, Azienda Azienda Unitaria Sanitaria Locale di Reggio Emilia , Reggio Emilia 42123, Italy Correspondence to: Alessia Ciarrocchi, email: Alessia.Ciarrocchi@ausl.re.it Keywords: papillary thyroid carcinomas; distant metastases; copy number alterations; next generation sequencing; TERT duplication Received: September 14, 2017 Accepted: October 27, 2017 Published: December 01, 2017 ABSTRACT Background: Papillary Thyroid Carcinomas (PTCs) are generally indolent tumors. However, a small but significant percentage of PTCs behaves aggressively, progressing to a diffuse metastatic spreading and leading to patient’s death. The lack of reliable markers for predicting the metastatic behavior of these tumors prevents a correct risk based stratification of the disease, thus contributing to the issue of patients’ overtreatment. In this study we aimed at identifying genetic features associated with the development of distant metastasis in PTCs. Results: We showed that DM PTCs are characterized by a moderate degree of copy number alterations but display low level of microsatellite instability and a low mutational burden. We identified duplication of Chr1q, duplication of Chr5p harboring the TERT genomic locus and mutations of TERT promoter as distinctive features of DM PTCs. These three genetic variables defined a signature (THYT1) that was significantly associated with a metastatic behavior and a shortened survival. We analyzed the THYT1 signature in PTCs fine needle aspirate biopsies (FNAB) and we demonstrating the applicability of this signature as a molecular marker in the pre-operative diagnostic setting of PTCs. Materials and Methods: A consecutive series of 2,937 thyroid malignancies, diagnosed at the Arcispedale S. Maria Nuova - IRCCS, Italy between 1978 and 2015 were searched to retrieve those who developed distant metastasis (DM, n = 50). We performed a deep profiling to explore the genomic landscape of these tumors. Conclusions: Overall our data identify the first genetic signature that independently predicts metastasis and negative outcome of PTCs, and lay the basis for the possible application of the THYT1 as prognostic marker to improve risk-based stratification and management of PTC patients.

Background: Clinical and molecular factors are essential to define the prognosis in patients with glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) methylation status, age, Karnofsky Performance Status (KPS), and extent of surgical resection are the most relevant prognostic factors. Our investigation of the role of gender in predicting prognosis shows a slight survival advantage for female patients. Methods: We performed a prospective evaluation of the Project of Emilia Romagna on Neuro-Oncology (PERNO) registry to identify prognostic factors in patients with GBM who received standard treatment. Results: A total of 169 patients (99 males [58.6%] and 70 females [41.4%]) were evaluated prospectively. MGMT methylation was evaluable in 140 patients. Among the male patients, 36 were MGMT methylated (25.7%) and 47 were unmethylated (33.6%); among the female patients, 32 were methylated (22.9%) and 25 were unmethylated (17.9%). Survival was longer in the methylated females compared with the methylated males (P = 0.028) but was not significantly different between the unmethylated females and the unmethylated males (P = 0.395). In multivariate analysis, gender and MGMT methylation status considered together (methylated females vs. methylated males; hazard ratio [HR], 0.459; 95% confidence interval [CI], 0.242-0.827; P = 0.017), age (HR, 1.025; 95% CI, 1.002-1.049; P = 0.032), and KPS (HR, 0.965; 95% CI, 0.948-0.982; P < 0.001) were significantly correlated with survival. Conclusions: Survival was consistently longer among MGMT methylated females compared with males. Gender can be considered as a further prognostic factor.

none 14 no Aim: To identify patients with recurrent glioblastoma after temozolomide (TMZ) concurrent with and adjuvant to radiotherapy who could benefit from TMZ rechallenge at the time of disease progression. Methods: We retrospectively evaluated 106 glioblastoma patients who had nonprogressive disease at first magnetic resonance imaging after completion of TMZ concurrent with and adjuvant to radiotherapy, a treatment-free interval (TFI) of at least 8 weeks and received TMZ rechallenge or a nitrosourea at the time of progression. Results: In patients with TFI ≥5 months, median survival was 17.7 and 11.6 months and median progression-free survival was 8.1 and 5.8 months in the TMZ and nitrosourea group, respectively. Longer TFI was associated with reduced risk for death (p = 0.002) and for disease progression (p = 0.005). Conclusion: TFI ≥5 months represents a predictor of retained TMZ sensitivity. none Franceschi, Enrico; Lamberti, Giuseppe; Visani, Michela; Paccapelo, Alexandro; Mura, Antonella; Tallini, Giovanni; Pession, Annalisa; De Biase, Dario; Minichillo, Santino; Tosoni, Alicia; Di Battista, Monica; Cubeddu, Alessio; Bartolini, Stefania; Brandes, Alba A* Franceschi, Enrico; Lamberti, Giuseppe; Visani, Michela; Paccapelo, Alexandro; Mura, Antonella; Tallini, Giovanni; Pession, Annalisa; De Biase, Dario; Minichillo, Santino; Tosoni, Alicia; Di Battista, Monica; Cubeddu, Alessio; Bartolini, Stefania; Brandes, Alba A*

IDH mutational status is required for proper diagnosis according to the WHO criteria revised in 2016. The single nucleotide polymorphism (SNP) rs11554137 (IDH1105GGT) at codon 105 of IDH1 has been reported in patients with several tumor types, including those with glioma. The aim of this study is to investigate the prevalence of IDH1105GGT in a cohort of brain tumors, and its association with clinicopathologic features and IDH1 and IDH2 missense mutations. Exon 4 of IDH1 and IDH2 was analyzed in a series of brain tumors classified according to current WHO criteria. DNA from control individuals was analyzed to infer the prevalence of IDH1105GGT in the reference population. Analysis was performed using next generation sequencing. IDH1105GGT was three times more frequent in patients with tumors (44/293 cases, 15.0%) vs. population controls (6/109, 5.5%) (p = 0.0102). IDH1105GGT was more frequent in grade III tumors (26.1%) compared to grade II (10.9%, p = 0.038) and grade IV tumors (13.7%, p = 0.041). IDH1 105GGT was more frequent in grade II and III tumors without an IDH tumor missense mutation (43.8%) than in those with (11.5%, p = 0.005). The IDH1105GGT SNP likely represents an important genetic marker, worthy of additional investigation to better understand the clinical and biological features of IDH-WT infiltrating gliomas.

Background Pancreatic adenocarcinoma (PDAC) is one of the deadliest human malignancies. Although surgery is currently the only effective treatment for PDAC, most patients survive less than 20 months after tumor resection. Objective The primary goal was to investigate alterations in KRAS, TP53, SMAD4 and CDKN2A/p16 in tumors from patients with exceptionally long survival after surgery. Methods Tumors from 15 patients with PDAC that survived more than 55 months after surgery ("LS") were analyzed for KRAS, TP53, IDH1, NRAS and BRAF using next-generation sequencing. SMAD4 and CDKN2A/p16 was tested using immunohistochemistry. MGMT promoter methylation was investigated. Results Tumors from "LS" have a lower prevalence of KRAS and TP53 mutations and had more frequently SMAD4 retained expression, if compared with that of patients died within 24 months from surgery. The survival of patients with wild-type KRAS and TP53 tumors was more than twice longer than that of patients bearing KRAS and TP53 mutations (90.2 vs. 41.1 months). Patients with KRAS wild-type tumors and that retained SMAD4 expression had a survival twice longer than cases with alterations in both genes (83.8 vs. 36.7 months). Eleven tumors (39.3%) showed MGMT methylation. Conclusions Our data indicate that absence of KRAS, TP53 and SMAD4 genetic alterations may identify a subset of pancreatic carcinomas with better outcome.

Background Epidermal Growth Factor Receptor (EGFR) molecular analysis is performed to assess the responsiveness to Tyrosine Kinase Inhibitors (TKIs) in patients with Non-Small Cell Lung Cancer (NSCLC). The existence of molecular intra-tumoral heterogeneity has been observed in lung cancers. The aim of the present study is to investigate if the percentage of mutated neoplastic cells within the tumor sample might influence the responsiveness to TKIs treatment. Material and methods A total of 931 cases of NSCLC were analyzed for EGFR mutational status (exon 18, 19, 20, 21) using Next Generation Sequencer. The percentage of mutated neoplastic cells was calculated after normalizing the percentage of mutated alleles obtained after next generation sequencer analysis with the percentage of neoplastic cells in each tumor. Results Next generation sequencing revealed an EGFR mutation in 167 samples (17.9%), mainly deletions in exon 19. In 18 patients treated with TKIs and with available follow-up, there was a significant correlation between the percentage of mutated neoplastic cells and the clinical response (P = 0.017). Patients with a percentage of mutated neoplastic cells greater than 56%, have a statistical trend (P = 0.081) for higher Overall Survival (26.3 months) when compared to those with a rate of mutated neoplastic cells lower than 56% (8.2 months). Conclusions The percentage of EGFR-mutated neoplastic cells in the tumor is associated with response to TKIs. A “quantitative result” of EGFR mutational status might provide useful information in order to recognize those patients which might have the greatest benefit from TKIs.

2028 Background: According to the 2016 WHO classification of Central Nervous System tumors, the assessment of exon 4 mutations in IDH1 or IDH2 genes is an essential step in the characterization of gliomas. The R132H mutation is the most frequent alteration in IDH1 gene, however other non-canonical IDH mutations have been identified. The aim of this study was to evaluate the prognostic role of IDH non-canonical mutations. Methods: We analyzed our institutional data warehouse for all consecutive patients (pts) with newly diagnosed, histologically proven grade II – IV IDH mutant gliomas. IDH sequencing was performed using the 454 GS-Junior next generation sequencer (NGS) (Roche Diagnostic, Mannheim, Germany). All analyses were performed on DNA from formalin fixed and paraffin embedded (FFPE) specimens. Results: The analysis included 493 pts with IDH mutations. We found 279 (56.6%) grade 2, 173 grade 3 (35.1%) gliomas, and 41 (8.3%) IDH mutant glioblastoma. Canonical IDH1 R132H mutation was found in 428 pts (86.8%). The remaining pts showed IDH2 (3.9%) or IDH 1 non-canonical mutations (mainly R132C, R132G, R132S – 9.3%). Median follow-up time was 80.5 months. Pts with non-canonical mutations showed a younger median age (32 vs 39 years, p < 0.001). Other clinical characteristics and treatments were similar across IDH groups. Median survival was 145 months (95%CI: 137.7 - 152.9) and 198.6 (95%CI 155.2– 242.1) in patients with IDH R132H and non-canonical mutations, respectively (p = 0.013). In multivariate analysis grading (p < 0.001), extent of surgery (p < 0.001), 1p19q codeletion (p = 0.003) and presence of non-canonical mutations (p = 0.022) showed a significant role for improved survival. Conclusions: Detecting non-canonical IDH1 mutations is essential for diagnosis and for prognosis in patients with gliomas. Differential enzymatic activity of non-canonical IDH1 mutations, resulting in different levels 2-hydroxyglutaratecould be the reason of improved survival.

2036 Background: The 2016 WHO classification dramatically changed the diagnosis of gliomas. Diffuse gliomas are classified according to the presence of IDH-mutation (IDH-mut) and the deletion of both 1p and 19q chromosome arms (1p/19q codel). Now debate is whether grade still has an independent prognostic value. The aim of this study was to find out if grade is a prognostic factor independently of molecular status. Methods: We analyzed our institutional data warehouse for all consecutive patients (pts) with newly diagnosed, histologically proven Grade II or Grade III IDH-mut gliomas. IDH 1/2 assessment by polymerase chain reaction (PCR)or immunohistochemistry (IHC) was accepted. Next Generation Sequencing (NGS) for IDH1(exon 4) and IDH2(exon 4) was performed on all specimens wild-type for the IDH. Results: The analysis included all the 399 pts who had a grade II (n = 250, 62.7%) or grade III (n = 149, 37.3%). Median follow-up time was 105.3 months. After surgery, 72 pts (18.0%) received RT alone, 44 (11.0%) received CT alone, 135 (33.8%) received both RT and CT, and 142 (35.6%) follow-up without any treatment. Median survival was 148.1 months. In multivariate analysis Grade (HR = 0.342, 95%CI: 0.221 – 0.531; P < 0.001) and 1p/19q codeletion (HR = 0.440, 95%CI: 0.290 – 0.668; P < 0.001) were independently associated with a lower risk for death. The difference in survival remained when adjusted for histological subtype. Residual disease after surgery or biopsy negatively affected survival (HR 2.151, 95%CI 1.375 – 3.367, P = 0.001). Post-surgical treatment with RT + adjuvant CT improves survival in respect to follow-up and other treatments (HR: 0.316, 95%CI 0.156 – 0.641, P = 0.001). Conclusions: Grade still affects survival in IDH mutant Grade II and III gliomas. This effect was independent onmolecular features, surgical extension and post-surgical treatments. Clinical management of gliomas should continue to take into account grade as well as molecular characteristics.

e14734 Background: IDH mutational status is required for diagnosis according to the 2016 WHO criteria. The single nucleotide polymorphism (SNP) rs11554137 ( IDH1105GGT) at codon 105 of IDH1 has been reported in patients with several tumor types, including glioma. The aim of this study is to investigate the prognostic role of IDH1105GGT Methods: We analyzed our institutional data warehouse for consecutive patients (pts) with newly diagnosed, histologically proven grade II or Grade III gliomas. IDH sequencing was performed using the 454 GS-Junior next generation sequencer (NGS) (Roche Diagnostic, Mannheim, Germany). All analyses were performed on DNA from formalin fixed and paraffin embedded (FFPE) specimens. Results: The analysis included 77 pts with grade II (n = 51, 66.2%) or grade III glioma (n = 26, 33.8%). Median follow up of this study was 91 months. Patients received biopsy/partial resection/complete resection in 7.8%, 70.1% and 22.1%, respectively. Postsurgical RT and/or chemotherapy was delivered in 64.9% of pts. IDH mutations affecting codons 132 (for IDH1) and 172 (for IDH2) were found in 71 pts (92.2%). IDH1105GGTwas found in 11 pts (14.3%), mainly harboring also IDH mutations (81.8%), and was more frequent in grade 3 (30.8%) than in grade 2 gliomas (5.9%, P = 0.006). Pts harboring both IDH mutations and IDH1105GGTshowed a significantly longer progression-free survival compared with pts with IDH mutation alone (78.7 months vs 49.9 months, p = 0.041). Conclusions: IDH1105GGT represents a promising novel biomarker in IDH mutated grade 2 and 3 gliomas and warrants further investigations.

The use of endoscopic ultrasonography has allowed for improved detection and pathologic analysis of fine needle aspirate material for pancreatic lesion diagnosis. The molecular analysis of KRAS has further improved the clinical sensitivity of preoperative analysis. For this reason, the use of highly analytical sensitive and specific molecular tests in the analysis of material from fine needle aspirate specimens has become of great importance. In the present study, 60 specimens from endoscopic ultrasonography fine needle aspirate were analyzed for KRAS exon 2 and exon 3 mutations, using three different techniques: Sanger sequencing, allele specific locked nucleic acid PCR and Next Generation sequencing (454 GS-Junior, Roche). Moreover, KRAS was also tested in wild-type samples, starting from DNA obtained from cytological smears after pathological evaluation. Sanger sequencing showed a clinical sensitivity for the detection of the KRAS mutation of 42.1%, allele specific locked nucleic acid of 52.8% and Next Generation of 73.7%. In two wild-type cases the re-sequencing starting from selected material allowed to detect a KRAS mutation, increasing the clinical sensitivity of next generation sequencing to 78.95%. The present study demonstrated that the performance of molecular analysis could be improved by using highly analytical sensitive techniques. The Next Generation Sequencing allowed to increase the clinical sensitivity of the test without decreasing the specificity of the analysis. Moreover we observed that it could be useful to repeat the analysis starting from selectable material, such as cytological smears to avoid false negative results.

Aims: In cystic and solid pancreatic lesions, KRAS mutational status refines the diagnosis of uncertain endoscopic ultrasound (EUS) aspirates. This test should have a fast turnaround time and ideally be performed at the centre where the patient is diagnosed. The Idylla KRAS Mutation Test enables standardisation even in units without molecular expertise. Methods: The Idylla test was designed for use with formalin-fixed paraffin-embedded (FFPE) sections. However, we directly pipetted 3 µL (corresponding to 1/10th of a DNA preparation from the aspirate sample) in the cartridge, which was automatically run as if an FFPE sample had been inserted. The performance was compared with Sanger sequencing, Allele Specific Locked Nucleic Acid PCR (ASLNAqPCR), and 454 Next Generation Sequencing (454-NGS) in light of clinicopathological end points. Results: Idylla yielded valid results in 49/52 (94.2%) cases, in 2 h. A total of 18/49 cases showed mutation either in KRAS exon 2 (14/18) or in exon 3 (4/18). Idylla KRAS test had 100% specificity and a sensitivity (55.1%) higher than Sanger sequencing (41.3%) and identical to ASLNAqPCR (55.1%). When the low-abundant mutant allele (

Background: The most appropriate management of recurrent glioblastoma is still controversial. In particular, the role of surgery at recurrence remains uncertain. Patients & methods: From our Institutional data warehouse we analyzed 270 consecutive patients who received second surgery for recurrent glioblastoma, to assess survival after second surgery, and to evaluate prognostic factors. Results: Complete resection was found in 128 (47.4%) and partial resection in 142 patients (52.6%). Median survival from second surgery was 11.4 months (95% CI: 10.0–12.7). Multivariate analysis showed that age (p = 0.001), MGMT methylation (p = 0.021) and extent of surgery (p < 0.001) are associated with better survival. Conclusion: A complete resection should be the goal for second resection and younger age and MGMT methylation status might be considered in the selection of patients.

Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs.RETmutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somaticRETmutations (RETmut group) and 34 with wild-typeRET(RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared withRETwt tumors,RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels inRETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis inRETmut MTCs may be more intense and complete than that found inRETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density,RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors.

The members of the PERNO Study Group were not individually captured in the metadata of the original publication. They are included in the metadata of this publication.

e14057Background: About 21-62% of GBM patients (pts) access to 3rd line therapy. In this setting, there is no defined standard. In this study we evaluated the outcome of pts who received 3rd line t...

AIM: To summarize the current knowledge about the potential relationship between hepatitis C virus (HCV) infection and the risk of several extra-liver cancers. METHODS: We performed a systematic review of the literature, according to the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) Statement. We extracted the pertinent articles, published in MEDLINE and the Cochrane Library, using the following search terms: neoplasm/cancer/malignancy/tumor/carcinoma/adeno-carcinoma and non-Hodgkin lymphomas, kidney/renal-, cholangio-, pancreatic-, thyroid-, breast-,oral-, skin-, prostate-, lung-, colon-, stomach-, haematologic. Case series, case-series with control-group, case-control, cohort-studies as well as meta-analyses, written in English were collected. Some of the main characteristics of retrieved trials, which were designed to investigate the prevalence of HCV infection in each type of the above-mentioned human malignancies were summarised. A main table was defined and included a short description in the text for each of these tumours, whether at least five studies about a specific neoplasm, meeting inclusion criteria, were available in literature. According to these criteria, we created the following sections and the corresponding tables and we indicated the number of included or excluded articles, as well as of meta-analyses and reviews: (1) HCV and haematopoietic malignancies; (2) HCV and cholangiocarcinoma; (3) HCV and pancreatic cancer; (4) HCV and breast cancer; (5) HCV and kidney cancer; (6) HCV and skin or oral cancer; and (7) HCV and thyroid cancer. RESULTS: According to available data, a clear correlation between regions of HCV prevalence and risk of extra-liver cancers has emerged only for a very small group of types and histological subtypes of malignancies. In particular, HCV infection has been associated with: (1) a higher incidence of some B-cell Non-Hodgkin-Lymphoma types, in countries, where an elevated prevalence of this pathogen is detectable, accounting to a percentage of about 10%; (2) an increased risk of intra-hepatic cholangiocarcinoma; and (3) a correlation between HCV prevalence and pancreatic cancer (PAC) incidence. CONCLUSION: To date no definitive conclusions may be obtained from the analysis of relationship between HCV and extra-hepatic cancers. Further studies, recruiting an adequate number of patients are required to confirm or deny this association.

Medulloblastoma is the most common central nervous system tumor in children, while it is extremely rare in adults. Multimodal treatment involving surgery, radiotherapy and chemotherapy can improve the prognosis of this disease, and recent advances in molecular biology have allowed the identification of molecular subgroups (WNT, SHH, Groups 3 and 4), each of which have different cytogenetic, mutational and gene expression signatures, demographics, histology and prognosis. The present review focuses on the state of the art for adult medulloblastoma treatment and on novel molecular advances and their future implications in the treatment of this disease.